Device and method for determining antibiotic susceptibility

ABSTRACT

The present invention provides a method of determining the antibiotic susceptibility of a microorganism comprising the following steps. First, a culture of the microorganism whose susceptibility is to be determined is admixed with an antibiotic to which susceptibility is to be assayed, and a permeabilizing agent for the microorganism present in a non-growth-inhibiting microorganism-permeabilizing effective amount to form an assay culture. Next, the assay culture is incubated under appropriate culture conditions and for a time sufficient to determine the susceptibility of the microorganism to the antibiotic. In another aspect, the present invention provides an improved method for antibiotic susceptibility testing of a microorganism in a culture by admixing the culture with an antibiotic to which susceptibility is to be assayed, and incubating the culture for a time sufficient to determine the susceptibility of the microorganism to the antibiotic, the improvement comprising admixing the culture with a permeabilizing agent for the microorganism present in a non-growth inhibiting microorganism-permeabilizing effective amount.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. DivisionalApplication Ser. No. 11/177,714 filed on Jul. 8, 2005 which is adivision application of U.S. Application Ser. No. 10/387,788, filed Mar.13, 2003, which claims priority of U.S. Provisional Application Ser. No.60/364,232, filed Mar. 13, 2002, all of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[Not applicable.]

BACKGROUND OF THE INVENTION

Clinicians and veterinarians often select antibiotic therapies forinfections on the basis of laboratory test results. The laboratorytests, known as antimicrobial or antibiotic susceptibility tests,determine the inhibitory activity of antibiotics against themicroorganisms that cause infections. If the antibiotic susceptibilitytest indicates that an antibiotic is sufficiently potent to treat aninfection, the microorganism causing the infection is reported to be“susceptible” to the antibiotic. If the test indicates a lack ofsufficient antimicrobial potency for successful therapy, themicroorganism is reported as “resistant” to the antibiotic. In sometests other categories of susceptibility may be reported, e.g. “moderatesusceptibility” or “intermediate susceptibility.”

A problem with currently available antimicrobial susceptibility tests istheir failure to reliably predict the outcome of therapy. Sometimes anantibiotic will fail to cure an infection even though the microorganismis susceptible to the antibiotic in the laboratory test. That is, thecurrent routine laboratory tests can be misleading and give anover-optimistic impression of the therapeutic potential of antibiotics.These tests can therefore cause patients to be given ineffectivetreatments. In serious infections, this inadequacy of current laboratorytests can have fatal consequences.

There are many reasons for failures of antibiotic therapies that wereinitiated on the basis of antibiotic susceptibility tests. Some involvepatient-related factors. Some involve pathogen-related factors. Howeverone explanation is error arising from a deficiency in the antibioticsusceptibility test itself. That deficiency is that current routineantibiotic susceptibility tests do not detect theantibiotic-inactivating potential of some microorganisms. Somemicroorganisms produce enzymes that inactivate antibiotics. The bestknown enzymes of this type are the β-lactamases that certain bacteriaproduce to inactivate β-lactam antibiotics. Such enzymes, which are notreliably detected in routine antibiotic susceptibility tests, may causesufficient antibiotic inactivation at the site of an infection in apatient to cause a treatment failure.

Plasmid-mediated AmpC β-lactamases were first reported in the 1980's.Bauernfeind, A., Y. Chong, and S. Schweighart 1989. Extendedbroad-spectrum β-lactamase in Klebsiella pneumoniae including resistanceto cephamycins. Infection. 17:316-321. They arose as a consequence ofthe transfer of chromosomal genes for inducible AmpC β-lactamases ontoplasmids. These enzymes have been reported in isolates of E. coli, K.pneumoniae, K. oxytoca, Salmonella spp., Citrobacter freundii,Enterobacter aerogenes, and Proteus mirabilis. The genes are typicallyencoded on large plasmids containing other antibiotic encodingresistance genes, leaving few therapeutic options. Although it has beenover ten years since plasmid-mediated AmpC β-lactamases were discovered,most clinical labs and physicians remain unaware of their clinicalimportance. As a result, plasmid-mediated AmpC β-lactamases often goundetected. Without detection, correct therapy for infected patients maynot be given.

Currently there are no NCCLS recommendations for detection ofplasmid-mediated AmpC β-lactamases. The three-dimensional test has beenused to detect these enzymes. However, this test is technicallydemanding and this has precluded its widespread adoption. Multiplex PCRis also currently available as a research tool for detection ofplasmid-mediated AmpC β-lactamases, but is not yet available as aroutine test for clinical labs. Perez-Perez FJ, Hanson ND 2002.Detection of plasmid-mediated AmpC beta-lactamase genes in clinicalisolates by using multiplex PCR. J Clin Microbiol. 40(6):2153-62.

The currently used antibiotic susceptibility tests, which measure onlythe antimicrobial activity of antibiotics and not the ability of themicroorganisms to cause antibiotic inactivation, fail to take intoaccount this very important determinant of the outcome of therapy. Thisdeficiency in the tests places clinicians at a disadvantage in selectingthe most appropriate antibiotics for their infected patients.

In summary, there is a need for antibiotic susceptibility tests thatprovide clinicians with information about both the antimicrobialactivity of antibiotics and, additionally, the ability of microorganismsto inactivate antibiotics. Such tests should improve the quality oftherapeutic decision-making by clinicians when selecting antibiotictherapies for patients with infections.

Antibiotic susceptibilities are determined routinely by either diskdiffusion or antibiotic dilution methods, or by methods that arederivatives of these two methods. In disk diffusion methods [for examplesee Bauer, A. W. et al., American Journal of Clinical Pathology.45:493-496 (1966); Bell, S. M., Pathology. 7:Suppl 1-48 (1975); Stokes,E. J., et al., Association of Clinical Pathologists Broadsheet, No. 55(revised) (1972)] a standard quantity of the causative microorganism isuniformly spread over the surface of an appropriate culture medium(hereafter referred to as agar). Then several filter paper disksimpregnated with specific amounts of selected antibiotics are placed onthe agar surface. The agar is then incubated for an appropriate periodat an appropriate temperature. During incubation the antibiotics diffuseout of the disks into the agar and the microorganism grows on thesurface of the agar, except in the areas where antibiotics inhibit itsgrowth. Inhibition of growth is detected as clear zones of no growth(inhibition zones) on the agar around the antibiotic disks. The sizes ofthe inhibition zones are measured and compared to establishedinterpretive criteria to determine the microorganism's susceptibility orresistance to antibiotics.

Dilution methods test antibiotic susceptibility on either solid agar orin liquid broth. [National Committee for Clinical Laboratory Standards1997. Methods for dilution antimicrobial susceptibility tests forbacteria that grow aerobically. Approved standard M7-A4. NationalCommittee for Clinical Laboratory Standards, Villanova, Pa.] In thedilution method a constant quantity of microbial inoculum is introducedinto a series of tubes or wells of broth (or onto one or more agarplates) containing varying concentrations of antibiotic. Afterincubation for an appropriate period the broth (or agar) tests areinspected and the lowest concentration of antibiotic that preventsdetectable growth of the microorganism recorded. This concentration isthe minimum inhibitory concentration (MIC) of the antibiotic.

Both disk diffusion and dilution methods are generally deficient in thatthey do not yield information about the ability of microorganisms toinactivate antibiotics.

Various techniques for detecting antibiotic-inactivating enzymes ofbacteria have been reported in the scientific literature. Sometechniques are used to detect the activity of specificantibiotic-inactivating enzymes (e.g. β-lactamases), while others arenon-specific and detect enzymes that inactivate more than one class ofantibiotics. The following are exemplary tests used to detectantibiotic-inactivating enzymes of bacteria.

Specific tests for the detection of chloramphenicol acetyltransferaseare reviewed in Chauchereau, A., et al., Analytical Biochemistry.188:310-316 (1990). These are complex tests to detect enzymaticinactivation of chloramphenicol and require special instruments capableof measuring the absorbance of light at specific wavelengths or ofmeasuring radioactivity. Such tests are not antibiotic susceptibilitytests and their complexity is such that they are unsuitable for routineclinical microbiology laboratories.

The production of β-lactamases by Staphylococcus aureus is inferred bythe production of a distinctive heaped-up margin of the inhibition zonearound a penicillin antibiotic disk. Gill, V. J., et al., J. Clin.Microbiol. 14:437-40 (1981). Beta-lactamase production by many types ofbacteria can also be detected chemically by testing the bacteria with anindicator substance such as nitrocefin. Oberhofer, T. R., et al., J.Clin. Microbiol. 15:196-9 (1982); O'Callaghan, C. H., et al., A.A.C.1:283-288 (1972). These tests are reliable indicators only ofβ-lactamase-determined resistance of Staphylococcus aureus,Staphylococcus epidermidis, Moraxella catarrhalis, Neisseria andHaemophilus species to certain types of penicillin antibiotics. They donot predict the potential for any other bacteria to resist thesepenicillins, and they do not predict the potential for any bacteria tobe resistant to any of the other classes of β-lactam antibiotics, suchas cephalosporins, cephamycins, monobactams, monocarbams, penems orcarbapenems. In short, these are useful tests of limited scope. Fortests of β-lactam antibiotics, a more comprehensive test is needed todetect the activities of all β-lactamases against all β-lactamantibiotics.

Disk diffusion tests can be modified by a pre-incubation procedure todetermine the ability of β-lactamases from Staphylococcus aureus toinactivate β-lactam antibiotics. Lacey, R. W., and A. Stokes, J ClinPathol. 30:35-9 (1977). This procedure results in smaller inhibitionzones than those for which the interpretive criteria of the tests werecalibrated. The preincubation procedure thereby invalidates theinterpretive tables that are necessary to determine antibioticsusceptibility or resistance. This is a serious deficiency because itwould be unethical to base therapy on this procedure which lacksvalidated interpretive criteria.

The clover leaf test [Andremont, A., et al., Proceedings ReunionInterdisciplinaire de Chimiotherapie Antiinfectieuse. Societe francaisede Microbiologie, Paris:50 (1982); Kjellander, J., et al., ActaPathologica Microbiologica Scandinavica. 61:494 (1964)] is a specialtechnique used to detect β-lactamases and is also claimed to detect twoother types of antibiotic-inactivating enzymes, chloramphenicolacetyltransferase and erythromycin esterase. This test is not anantibiotic susceptibility test and must be set up as an additionalprocedure. This is inconvenient and therefore a disadvantage.Furthermore there are doubts about the validity of results obtained withthis procedure. Jorgensen, P. E., Chemotherapy. 31:95-101 (1985); Reig,M., et al., European journal of Clinical Microbiology. 3:561-562 (1984).

The cefoxitin induction test [Sanders, C. C., and W. E. Sanders, Jr.,A.A.C. 15:792-797 (1979)] is a special procedure for detecting aparticular type of bacterial β-lactamase, the inducible AmpC β-lactamaseof Bush Group 1. Bush, K., et al., A.A.C. 39:1211-1233 (1995). This testdoes not detect all types of β-lactamases, and like the clover leaf testit is a special procedure that can be used to supplement antibioticsusceptibility tests. It is not, in itself, an antibiotic susceptibilitytest.

The double disk potentiation test (and its derivatives) involvesstrategically placing an amoxicillin/clavulanate orticarcillin/clavulanate disk 20 to 30 mm from disks containingcefotaxime, ceftriaxone, ceflizoxime, ceftazidime, cefepime or aztreonamon an agar plate. It is therefore possible to determine if a strain ofEnterobacteriaceae produces a special type of β-lactamase known as anextended-spectrum β-lactamase. Brun-Buisson, C., et al., Lancet.ii:302-306 (1987). The test is based on the ability of the β-lactamaseinhibitor, clavulanate, to inhibit the extended-spectrum β-lactamase andprevent it from inactivating the cephalosporin or aztreonam antibioticsin the test. This is a special procedure, not a routine antibioticsusceptibility test, and detects only certain types of β-lactamases. Itis therefore inconvenient and limited in scope.

A variety of disk and dilution tests have been derived from theprinciple of the double risk test. Brown, D. F., et al., J. Antimicrob.Chemother. 46:327-8 (2000); Cormican, M., G., et al., JCM. 34:1880-1884(1996); Ho, P. L., et al., JAC. 42:49-54 (1998); Moland, E. S., et al.,JCM. 36:2575-9 (1998); Sanders, C. C., et al., JCM. 34:2997-3001 (1996);Schooneveldt, J. M., et al., Pathology. 30:164-8 (1998); Thomson, K. S.,et al., Antimicrob. Agents Chemother. 43:1393-400 (1999). That is, theyuse the ability of a β-lactamase inhibitor to inhibit anextended-spectrum β-lactamase to detect this type of β-lactamase.

The 3-dimensional test [Thomson, K. S., et al., J. Antimicrob.Chemother. 13:45-54 (1984); Thomson, K. S., and C. C. Sanders, A.A.C.36:1877-1882 (1992); U.S. Pat. No. 5,466,583] is an approach thatpartially fulfills the need for improved antibiotic susceptibilitytesting.

In performing the direct form of the 3-dimensional test a standardquantity of the causative microorganism is uniformly spread over thesurface of an agar plate in the usual manner for performing a diskdiffusion test. However, before placing the antibiotic disks onto thesurface of the agar, the 3-dimensional inoculation is performed. This iseffected by using a sterile scalpel to cut a slit in the agar 3 mm toone side of where the antibiotic disks will be placed. A dense liquidinoculum of the test microorganism is then dispensed into the slit, theantibiotic disks are placed on the agar 3 mm from the slit, and the testis incubated.

After incubation the inhibition zones are measured by standardprocedures to determine the susceptibility or resistance of themicroorganism to the test antibiotics according to the interpretivecriteria of the disk diffusion test. However, in addition to this,enzymatic inactivation of the antibiotics can be detected by inspectingthe intersections of the 3-dimensional inoculum with the margins of theinhibition zones. Antibiotic inactivation results in a distortion ordiscontinuity in the usually circular inhibition zone. (Thesedistortions or discontinuities are hereafter referred to as“3-dimensional effects”.)

The 3-dimensional test thus allows the laboratory to report to theclinician not only the susceptibility or resistance of a microorganismto antibiotics, but also the ability of the microorganism to inactivatethe antibiotics. As a hypothetical example, whereas a conventionalantibiotic susceptibility test might indicate that a microorganism wassusceptible to the two antibiotics, cefaclor and cefoxitin, the3-dimensional test might provide additional information to show that themicroorganism produced an enzyme capable of inactivating cefaclor butnot cefoxitin. Thus, although the conventional test indicated that bothantibiotics appeared to be equally efficacious, it would appear, fromthe additional information provided by the 3-dimensional test, that onlycefoxitin might not be inactivated in the patient and therefore wouldconstitute a more effective treatment than cefaclor. In this example,the information provided by the 3-dimensional test could assist aclinician to make a better choice of therapy.

In addition to the direct form of the 3-dimensional test, the indirectform is used for testing microorganisms when inhibition zones are smallor absent, or as a research or diagnostic method. The indirect test isperformed by inoculating the surface of the agar with a fullysusceptible assay strain such as Escherichia coli ATCC 25922. Afterthis, the 3-dimensional slit is cut in the agar and inoculated with asuspension of the test microorganism. Although the indirect testprecludes the simultaneous determination of antibiotic susceptibilities,it permits investigation of the antibiotic inactivating enzymes ofmicroorganisms for which the inhibition zones are too small to yield3-dimensional results when the test is performed in the direct form ofthe test.

There are several problems with the 3-dimensional test. These problemsinclude the following:

a. The procedure for making the slit in the agar for the 3-dimensionaltest is inconvenient and technically difficult to perform correctly.

b. Making the slit is potentially dangerous to laboratory staff becausea scalpel blade contaminated with pathogenic bacteria is an infectionhazard.

c. It is also technically difficult to accurately deliver the liquid3-dimensional inoculum into the slit without overfilling the slit andpossibly invalidating the test.

A variety of chemicals have been reported to disrupt or permeabilizemicrobial membranes, thereby increasing their permeability and causingloss of cellular contents. These include certain antibiotics,detergents, chelating agents, polycations, hydrophobic dyes, andenzymes. Nikaido, H., and M. Vaara, Microbiol Rev. 49:1-32 (1985);Piers, K. L., et al., Antimicrob. Agents Chemother. 38:2311-2316 (1994).These chemicals are often bacteriostatic and/or bacteriocidal in normaluse.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of determiningthe antibiotic susceptibility of a microorganism comprising thefollowing steps. First, a culture of the microorganism whosesusceptibility is to be determined is admixed with an antibiotic towhich susceptibility is to be assayed, and a permeabilizing agent forthe microorganism present in a non-growth-inhibitingmicroorganism-permeabilizing effective amount to form an assay culture.Next, the assay culture is incubated under appropriate cultureconditions and for a time sufficient to determine the susceptibility ofthe microorganism to the antibiotic.

Preferably, the permeabilizing agent is dissolved or dispersed in acarrier. The carrier can be a solid carrier. A preferred solid carrieris a paper disk. Alternatively, the carrier can be a liquid carrier.

In one embodiment, the permeabilizing agent is a buffer solution. Apreferred buffer solution is Tris/EDTA.

In another embodiment, the culture is provided on a solid growth medium.Alternatively, the culture is provided in a liquid growth medium.

In a further embodiment, the carrier is a solid carrier, the culture isprovided on a solid growth medium, the antibiotic is provided on a solidsupport, and the admixing is done by contacting the culture with thesolid carrier and the antibiotic provided on a solid support.

In another aspect, the present invention provides an improved method forantibiotic susceptibility testing of a microorganism in a culture byadmixing the culture with an antibiotic to which susceptibility is to beassayed, and incubating the culture for a time sufficient to determinethe susceptibility of the microorganism to the antibiotic, theimprovement comprising admixing the culture with a permeabilizing agentfor the microorganism present in a non-growth inhibitingmicroorganism-permeabilizing effective amount.

Preferably, the permeabilizing agent is a buffer solution. A preferredbuffer solution is Tris/EDTA.

In one embodiment, the permeabilizing agent is dissolved or dispersed ina carrier. The carrier can be a solid carrier. A preferred solid carrieris a paper disk. Alternatively, the carrier can be a liquid carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a close up of a zone of distortion surrounding a cefoxitindisk.

FIG. 2 shows one embodiment of the invention. Here, the indirect form ofa method of the invention is shown in which five test microorganisms anda negative control organism are tested. In this assay the fullysusceptible strain E. coli ATCC 25922 was used as the lawn culture toassay for inactivating factors produced by the test and controlmicroorganisms. Three commercially manufactured cefoxitin disks (labeledFOX 30) are bracketed by the reagent test disks. A test or controlorganism was applied to each of the reagent disks prior to theirplacement proximal to the cefoxitin disks. Strong indentations of theinhibition zone margins indicate inactivation of cefoxitin byinactivating factors released from the test microorganisms subsequent toencountering the permeabilizing agent in the disks. The negative controlhas no distortion (bottom disk of top set of 3 disks with orientationpointing to 12 o'clock). A weakly positive test occurred at the diskclosest to 9 o'clock (shown as slight flattening or blunting of theinhibition zone margin). This result was obtained with a strain ofKlebsiella pneumoniae previously determined to be a low level producerof a cefoxitin-inactivating β-lactamase. (This strain is a potentialcandidate as a quality control strain associated with the threshold ofsensitivity of detection of the test for this particular antibioticinactivating factor).

FIG. 3 shows a three-dimensional test demonstrating the enzymaticinactivation of cefoxitin by a penicillin-resistant strain ofKlebsiella.

FIG. 4 shows an embodiment of the invention as described above for FIG.2, except that only a single cefoxitin disk is shown. The portion ofFIG. 4 marked “Positive” displays the distorted inhibition zonecharacteristic of a permeabilizing agent of the invention. The portionof FIG. 4 marked “Negative” displays the lack of distortion where acontrol reagent disk, not having a permeabilizing agent, is placedproximal to the antibiotic disk.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The invention provides a novel method of determining the antibioticsusceptibility of a microorganism based upon increasing the permeabilityof the cell wall and/or cell membrane(s) of a microorganism byincubating a microorganism in the presence of a permeabilizing agent.This permeabilizing agent is preferably present in an amount thatneither kills the microorganism nor significantly inhibits its abilityto replicate, but nevertheless permeabilizes the microorganism. Whilenot wishing to be bound by theory, it is believed that the presence ofan appropriate concentration of permeabilizing agent permits theenhanced liberation or release of antibiotic-inactivating factors (e.g.,enzymes) from the microorganism. The liberation or release of suchantibiotic-inactivating factors permits a microorganism to grow in thepresence of the antimicrobial agent that is inactivated by the releasedfactors.

In another aspect, the invention provides a novel method of determiningthe presence of antibiotic-inactivating factors, such asantibiotic-inactivating enzymes, comprising the following steps. First,a culture of the microorganism which is suspected of producing orliberating antibiotic-inactivating factors is admixed with an antibioticto which the antibiotic-inactivating factors are capable ofinactivation, and a permeabilizing agent for the microorganism presentin a non-growth-inhibiting microorganism-permeabilizing effective amountto form an assay culture. Next, the assay culture is incubated underappropriate culture conditions and for a time sufficient to determinethe presence of antibiotic-inactivating factors produced or liberated bythe microorganism capable of inactivating the antibiotic.

It has been found that a permeabilizing agent can be used to detectantibiotic-resistant microorganisms that would otherwise register asfalsely sensitive to a test antibiotic, by facilitating the liberationor release of antibiotic-inactivating factors. Surprisingly, themicroorganism can grow in the presence of a permeabilizing agent that isnormally lethal to a microorganism, or inhibits the growth of amicroorganism.

In one aspect, the invention comprises the use of a permeabilizingagent, in a non-growth-inhibiting, microorganism-permeabilizingeffective amount. As set forth in more detail elsewhere herein,determining a non-growth-inhibiting amount of a permeabilizing agent isa matter of routine experimentation involving the titration of thepermeabilizing agent for its ability to significantly inhibitmicroorganism growth. Similar, the determination of amicroorganism-permeabilizing effective amount of a permeabilizing agentis a matter of routine experimentation involving the titration of thepermeabilizing agent for its ability to permeabilze a microorganism,such as by liberation of antibiotic-inactivating factors from a knownantibiotic-resistant strain of a test microorganism.

The permeabilizing agent, in the amount set forth herein, is admixedwith a culture of a microorganism whose susceptibility to an antibioticis to be determined. Alternatively, the admixture is made to determinethe micoorganism's capacity to generate or liberate one or moreantibiotic-inactivating factors. The culture is prepared according tomethods that are well known in the art, such as by introducing aninoculum of the microorganism (such as from a clinical sample) into anappropriate growth medium. The growth medium can be a liquid growthmedium, such as Luria or Meuller-Hinton broths, or a sold growth medium,such as an agar plate supplemented with necessary nutrients, such as aMueller-Hinton agar plate.

Once the culture is prepared, the microorganism is admixed with one ormore antibiotics (antimicrobial agents) to be assayed. In the case ofassaying the microorganism for its ability to make or excrete anantibiotic-inactivating factor, the presence of the antibiotic isrequired both to provide a control for the assay, and to facilitate theproduction or liberation of the inactivating factor itself. Theantibiotic can be needed to initiate or enhance the production of theantibiotic-inactivating factor by the microorganism. In the absence ofthe antibiotic, the microorganism does not necessarily need to make,maintain, sequester, or liberate the antibiotic-inactivating factor.

Antibiotic-susceptibility assays are set forth elsewhere herein, and arenot meant to be limiting with respect to such assays that can be adaptedfor use in the instant invention. The culture is also admixed with thepermeabilizing agent.

If needed, a control culture, without either antibiotic orpermeabilizing agent, or both, can be prepared under similar conditions.Where the admixture is done with a liquid growth medium and apermeabilizing agent in a liquid carrier, as described elsewhere herein,serial dilutions of the antibiotic to be assayed are prepared in theliquid growth medium. Parallel samples are admixed with or without thepermeabilizing agent in the liquid carrier. An antibiotic that at aparticular antibiotic dilution shows antimicrobial activity in theabsence of permeabilizing agent, but is ineffective at that dilution inthe presence of permeabilizing agent, would be considered ineffectiveagainst the assayed microorganism, on the basis that the microorganismliberates a factor that inactivates that antibiotic.

The thus prepared culture is then incubated under appropriate cultureconditions and for a time sufficient to determine the susceptibility ofthe microorganism to the antibiotic or antibiotics of interest. Forexample, an agar plate can be incubated at 35 degrees Celsius for 12 to18 hours in an environmentally controlled incubator. A culture in aliquid medium can be incubated in a shaking water bath at 35 degreesCelsius for 12 to 18 hours on a bench top. The appropriate cultureconditions and sufficient times are well known to workers of ordinaryskill in the art.

As noted, the permeabilizing agent can be provided in a liquid or asolid carrier, dispersed or dissolved therein. For example, in the caseof a liquid permeabilizing agent, such as a buffer solution, the buffersolution is itself in a liquid carrier. Alternatively, a detergent canbe dispersed in an aqueous medium, or in growth broth, for use in themethods of the invention.

In another aspect, the permeabilizing agent is provided on a solidsupport. For example, the buffer solution can be impregnated onto asolid support such as a piece of filter paper. The filter paper is thenleft to dry, and the permeabilizing agent is dispersed onto the solidcarrier.

In one embodiment, the impregnated disk is used in an antibioticsensitivity assay to permeabilize the microorganism whose antibioticsensitivity is being measured, without killing or significantlyinhibiting the growth of the microorganism. An appropriate amount(concentration) of a permeabilizing agent to be used in preparing theseimpregnated disks can be determined to effectuate liberation ofantibiotic-inactivating factor(s) from a microorganism, without killingor inhibiting the growth of a microorganism.

The invention is designed to increase the amount of information providedby laboratory tests in which the susceptibility of microorganisms toantimicrobial agents (sometimes referred to herein as antibiotics) isevaluated by disk diffusion methods or by other antibiotic-sensitivitydetermining methods. The invention provides a means to routinely detectthe ability of microorganisms to inactivate antibiotics. The informationcan be useful in selecting appropriate antiinfective therapies for thetreatment of infections, in providing epidemiological information aboutmicroorganisms, in antibiotic research, and in other fields ofbiological research such as genetics and enzymology.

In a preferred embodiment, the invention comprises providing a filterpaper disk impregnated with an agent such as a chemical that willfacilitate the release of antibiotic inactivating enzymes from bacteriaor other microorganisms. This filter paper disk impregnated with thechemical is sometimes referred to herein as the reagent disk. Typicallythe agent in the reagent disk will be a permeabilizing agent that willdisrupt the outer membrane, or both the outer and cytoplasmic membranesof a microorganism such as a bacterium to releaseantibiotic-inactivating enzymes from within the microorganism. In apreferred method of the invention, a disk impregnated with apermeabilizing agent is provided.

A suitable growth-medium-containing agar plate is inoculated with a lawnof test microorganism (e.g., a clinical sample, or a strain of bacteriafor which antibiotic sensitivity is to be determined). Anantibiotic-impregnated disk is placed onto the lawn of microorganism.The test microorganism is additionally inoculated onto a providedreagent disk, and that reagent disk is placed on the agar adjacent to,but not in contact with, an antibiotic disk of interest. The agar plateis then incubated according to methods well known in the art. Afterincubation, the results are interpreted as follows. Enzymaticinactivation of an antibiotic can be detected by inspecting the marginof the inhibition zone in the vicinity of the reagent disk. Antibioticinactivation results in a distortion in the usually circular zone ofinhibition. The presence of this distortion indicates that the testmicroorganism is capable of producing antibiotic-inactivating factors.From a clinical perspective, the use of an antibiotic that exhibits azone of distortion would be contraindicated for treatment of aninfection with the test microorganism.

It should be understood that while a preferred embodiment of theinvention is as described above, the invention is not limited to thatembodiment. Other solid carriers other than a filter paper disk, such asfilter paper strips, pieces of nitrocellulose, or other carriers couldbe used as vehicles to effect contact between the microorganism and thereagent. Test methodologies other than the disk diffusion test, such asthe E test [Brown, D. F. J., and L. Brown, J. Antimicrob. Chemother.27:185-190 (1991)], or the dilution methods can also be modified fordetection of antibiotic inactivating enzymes without departing from thespirit and scope of the invention.

In another aspect, the invention provides an improved method forantibiotic testing of a microorganism in a culture, as exemplified bythe antibiotic susceptibility testing assays discussed elsewhere herein.The improvement comprises admixing the culture with a permeabilizingagent of the invention, in an amount that does not significantly inhibitthe growth of the culture but is effective in permeabilizing themicroorganisms.

In a further embodiment, the invention comprises an indirect form fortesting microorganisms when inhibition zones are small or absent, or asa research or diagnostic method. The indirect form of test is performedby inoculating the surface of the agar with a fullyantibiotic-susceptible assay strain such as Escherichia coli ATCC 25922.After this, the provided reagent disk is inoculated with a suspension ofthe test microorganism (e.g., a clinical isolate) and the test performedas described above except that the assay strain will grow on the surfaceof the agar instead of the causative organism. Although the indirectform of a method of the invention can preclude the simultaneousdetermination of antibiotic susceptibilities, it permits investigationof the antibiotic inactivating enzymes of microorganisms for which theinhibition zones are too small to yield results when the test isperformed in the direct form of the test.

Exemplary permeabilizing agents useful in the present invention includethose agents that increase the permeability of the cell wall and/or cellmembrane(s) of a microorganism, but that are present in amounts orconcentrations that will not significantly inhibit the growth orreproduction of the assayed microorganism. As is well known to those ofordinary skill in the art, not all microorganisms possess both a cellwall and a cell membrane; moreover, those microorganisms that possessboth a cell wall and a cell membrane do not have walls or membranes thatare structurally identical. For example, bacteria are broadlycategorized as Gram-positive or Gram-negative based upon their cellwall/cell membrane structure. A permeabilizing agent useful in thepresent invention preferably permeabilizes the cell surface structure(cell wall or cell membrane, or both) such that antibiotic-inactivatingfactors are liberated from the cell. However, it is also preferred thatthe permeabilizing agent not kill or significantly inhibit the growth ofthe microorganism.

Specific exemplary permeabilizing agents include the following:

Inorganic salts, such as sodium chloride (NaCl), magnesium chloride(Mg₂Cl), potassium chloride (KCl) and the like;

Quaternary ammonium compounds such as benzalkonium chloride (BAC),cetylpyridium chloride, 3-(trimethoxysilyl)propyldimethyloctadecylammonium chloride and the like;

Buffer solutions, such as Tris/EDTA (TE) buffer, NaCl/Tris/EDTA (STE)buffer, glucose/Tris/EDTA (GTE) buffer, and the like;

Hypotonic or hypertonic agents, such as sugar solutions, includingglucose, dextrose, sucrose, fructose, lactose and the like;

Antibiotics such as Piperacillin, Aztreonam, Amdinocillin, Ceftazidime,Polymyxin B, Polymyxin B nonapeptide, or Gentamicin, and the like;

Commercially available detergent or surfactant-containing agents such asBlind Brite™, Maxaquin™, HyVee Glass Cleaner™, Life Tree™, Rave™, Dawn™,and the like;

Other commercially available materials such as Isoclean™ concentrate,Sight Savers™ lens cleaner, Wash™ (green handwash), Micro Lab CleaningSolution™, Jet Clean™ test tube cleaner, Limonene™, and the like;

Other surfactants and/or detergents such as TritonX-100, sodium dodecylsulfate (SDS), SDS (5%) mixed with acetone, Sarcosyl™, Sarcosyl™ mixedwith acetone, Tween™ (polysorbate) 80, Tween™ 80 mixed with acetone,Tween™ 85, Brij™ 56, polyethylene glycol, polypropylene glycol,dansyl-polymyxin and the like.

Other exemplary permeabilizing agents include CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)electrophoresis reagent, ≦10 mM (Sigma cat # C-9426), lactic acid, andsodium hexametaphosphate (sodium polyphosphate) (chelating agent).

Natural peptide agents such as cecropins (cationic peptides), melittin(cationic peptide), bactenecin, magainins (frog host defense peptides),tachyplesins (cationic peptides), polyphemusins (cationic peptides), andsynthetic peptides and the like can also be used as preferredperneabilizing agents.

A preferred permeabilizing agent is Tris/EDTA (TE) buffer, commerciallyavailable from Sigma-Aldrich (catalog number T-9285). As supplied, theTE buffer is at a 100× concentration, comprised of 1.0 molar Tris-HCl(pH approximately 8.0) and 0.1 molar EDTA. The TE buffer is preferablyplaced onto a disk at a concentration of 100 millimolar, 200 millimolar,0.5 molar, 1 molar, 2 molar or 10 molar. The TE buffer is furtherpreferably placed onto a disk at a concentration of 100×, 50×, 25× and10×. Methods for determining the optimal concentration of a particularpermeabilizing agent are given elsewhere herein.

As noted elsewhere herein, a permeabilizing agent preferably permits orfacilities the release of antibiotic-inactivating factors from within amicroorganism while not significantly inhibiting the growth of thatmicroorganism. The ability of a permeabilizing agent to liberate orrelease antibiotic-inactivating factor(s) from a microorganism can bedetermined by, for example, quantitation of the amount of antibioticinactivating factor, such as an enzyme, released. Such quantitation canbe determined by, for example, spectrophotometric hydrolysis assaysusing either nitrocefin or cefaclor as substrates. The ability of apermeabilizing agent to liberate or release antibiotic-inactivatingfactor(s) from a microorganism can also be determined by, for example,using the methods of the present invention, or using otherantibiotic-sensitivity tests such as the 3-dimensional test discussedelsewhere herein. Exemplary determinations of the ability of apermeabilizing agent to liberate or release antibiotic-inactivatingfactor(s) from a microorganism are described in the Examples set forthelsewhere herein.

A preferred permeabilizing agent also does not significantly inhibit thegrowth of a microorganism. This property of a preferred permeabilizingagent can be determined by, for example, applying varying concentrationsof a particular permeabilizing agent to lawn cultures of a variety ofmicroorganisms to determine if inhibition of growth of the lawn cultureoccurred. Alternatively, the growth rates of various microorganisms inthe presence of varying concentrations of a permeabilizing agent canalso be determined, using techniques well known in the art. Exemplarydeterminations of the ability of a permeabilizing agent to permit thegrowth of a microorganism are described in the Examples set forthelsewhere herein.

In another embodiment, the present invention comprises a kit comprisinga permeabilizing agent-impregnated disk and instructions for performingan antibiotic sensitivity test to detect microorganisms that liberateantibiotic-inactivating factors. In a preferred embodiment, thepermeabilizing agent is Tris/EDTA buffer.

As can be readily seen, the invention provides novel benefits andadvantages over other antibiotic-sensitivity testing methods. Theinvention permits antibiotic-sensitivity testing to be performed, forexample, on the surface of the agar, rather than the technicallychallenging aspect of some other methods that require the insertion ofbacterial inoculum into a slit in the agar. Furthermore, the inventiondiscloses the use of a permeabilizing agent that facilitates the releaseof antibiotic inactivating factors from microorganisms, withoutsignificantly inhibiting the growth of those microorganisms. Currentantibiotic susceptibility testing techniques cannot enhance the releaseof such factors, leading to the possibility of false-positive results.Therefore, current antibiotic susceptibility testing techniques can leada clinician to believe that a particular microorganism is sensitive toan antibiotic based on these in vitro results, only to find empiricallythat the antibiotic is ineffectual in vivo. The present inventionprovides more robust in vitro information that can detect microorganismsthat are likely to be resistant to a particular antibiotic in vivo.

EXAMPLES Example 1 Determination of Release of Antibiotic-InactivatingFactors

In this Example, E. coli MISC 208 (which produces the extended spectrumβ-lactamase SHV-2) was used as a test organism and nitrocefin was thesubstrate in spectrophotometric hydrolysis assays. The E. coli strainwas harvested directly into 100 microliters of Mueller Hinton Broth andmixed with 100 microliters of the permeabilizing agent.

In the hydrolysis assay, units of activity are calculated as:${Units} = \frac{{Change}\quad{in}\quad{optical}\quad{density}\quad({OD})\text{/}{minute}\quad \times 10}{{Extinction}\quad{coefficient}}$

The hydrolysis assay was performed essentially as described byO'Callaghan, C. H., et al., A.A.C. 1:283-288 (1972). Briefly, a 100 μMsolution of nitrocefin was used as a substrate. The nitrocefin wasprewarmed to 37° C. The hydrolysis assay was performed at 37° C. at awavelength of 389.5 with an extinction coefficient of −0.024. Anappropriate cuvette was filled with 0.9 ml of the prewarmed nitrocefinsubstrate and 100ul of permeabilized bacterial suspension was added. Thecontents of the cuvette were immediately mixed by inversion and thecuvette was placed in a spectrophotometer. Absorbance was assayed at5-second intervals for up to 5 minutes with the 37° C. temperaturemaintained by means of a circulating water bath that warmed the cuvetteblock. When the assay was complete, the activity was calculatedaccording to the above formula. Units of β-lactamase ActivityPermeabilizing Agent (Nitrocefin as substrate) TE/Room temp 47 TE/0° C.102 Tris/0° C. 41 Water (control) 0

Example 2 Determination of Release of Antibiotic-Inactivating Factors

The experiment in Example 1 was repeated using E. coli MISC 208 andcefaclor as substrate in the hydrolysis assays. Units of β-lactamaseActivity Permeabilizing Agent Preparation (Cefaclor as substrate)Benzalkonium chloride 200 μg/ml 16 Polymyxin B 10 μg/ml 3.8 Polymyxin B30 μg/ml 15 TE 79

Example 3 Determination of Release of Antibiotic-Inactivating Factors

The experiment in Example 1 was repeated using E. coli V1104 to evaluatea variety of permeabilizing agents. Nitrocefan was the hydrolysissubstrate. Units of β-lactamase Activity Permeabilizing AgentPreparation (Nitrocefin as substrate) Aztreonam 210 No reagent/0° C. 214No reagent/45° C. 0 Ceftazidime 196 Piperacillin 202

Other exemplary microorganisms that can be used in the determination ofrelease of antibiotic-inactivating factors include those in thefollowing Table. Strain Enzyme Units (vs nitrocefin) E. coli V1104 202E. coli 165* pI 5.95 TEM ESBL 43 MG32 TEM-1 + TEM-12 349 K. pneumoniaeV1102 227

Example 4 Determination of Lack of Significant Growth Inhibition

A variety of test strains have been harvested directly into 100microliters of Mueller Hinton Broth and mixed with 100 microliters ofeach permeabilizing agent of interest. Then 20 microliters of each ofthese mixtures was applied to blank filter paper disks, and the diskstransferred to freshly inoculated agar lawn cultures of a variety oftest microorganisms and the plates were incubated overnight at 37° C.After overnight incubation, the cultures were inspected for the presenceor absence of an inhibition zone around the disk. An inhibition zoneinferred unsuitability of the permeabilizing agent for the test at theconcentration used.

A variant of this method is to directly apply the reagent to a diskwithout previously mixing the reagent with a microorganism, and toevaluate as above.

Making the reagent disk

-   -   1. A solution of equal parts of physiological saline and Tris        EDTA (Sigma Chemicals) is made.    -   2. A 20 microliter aliquot of the permeabilizing agent solution        was placed onto a filter paper disk and allowed to dry.

Performing and Interpreting the Test

-   -   1. The filter paper disk impregnated with the permeabilizing        agent was rehydrated with 20 microliters of sterile        physiological saline.    -   2. Using a cotton swab or loop, a test organism was rubbed onto        the rehydrated disk.    -   3. A Mueller Hinton Agar plate was inoculated with a 0.5        McFarland of E. coli ATCC 25922.    -   4. A cefoxitin disk was placed onto the surface of the agar        plate. The rehydrated disk inoculated with the test organism        next was placed next to the cefoxitin disk, at a distance of        about 0.5-1.0 mm.    -   5. The agar plate was incubated at 37° C. for about 16 hours, or        overnight.    -   6. Distortion of the cefoxitin zone was interpreted as a        positive result (i.e., the presence of an        antibiotic-inactivating factor was detected), whereas lack of        distortion of the cefoxitin zone was interpreted as a negative        result. See FIGS. 1 and 2.

Example 5 Detection of Antibiotic-Resistant Microorganisms

This study reports its utility for investigation of Klebsiella isolatessuspected of harboring pAmpCs.

Methods: Thirty isolates of Klebsiella spp. with cefoxitin MICs ≧16micrograms per milliliter were investigated using the methods of theinvention. All negative results were confirmed with thethree-dimensional test, discussed elsewhere herein, and positive resultswere confirmed by isoelectric focusing (IEF) and inhibitor studies. Afilter paper disk was impregnated with 100×TE which had been dilutedwith an equal volume of physiological saline. The disk was rehydratedwith 20 microliters of saline and several colonies of the test isolatewere inoculated onto the disk. The inoculated disk was then placedbeside a cefoxitin disk (almost touching) on a Mueller-Hinton agar plateinoculated with a lawn of E. coli ATCC 25922. After overnightincubation, a positive test appeared as a flattening or indentation ofthe cefoxitin inhibition zone near the test disk. A negative test had anundistorted zone.

Results: Of the 30 isolates tested, 10 were positive and 20 werenegative (also negative with the three-dimensional test). IEF andinhibitor studies of the positive isolates indicated that 9 of the 10isolates produced β-lactamases that were inhibited by cloxacillin withpIs of either 7.2, 7.7 or ≧9.0, findings consistent with AmpCproduction. These enzymes were interpreted to be plasmid-mediatedbecause Klebsiella lacks a chromosomal AmpC. The other positive isolatehad an elevated imipenem MIC and produced a carbapenem-hydrolyzingenzyme that also hydrolyzed cefoxitin.

Materials and Methods

Isolates

Thirty recent isolates of Klebsiella spp. were collected from patientsin United States hospitals.

Susceptibility

Antimicrobial susceptibility was determined by NCCLS microdilutionmethodology using an investigational Microscan dehydrated panel.

Three-dimensional test

Enzymatic inactivation of cefoxitin was investigated by thethree-dimensional method. The surface of a Mueller-Hinton agar plate wasinoculated with a lawn of a standardized inoculum of E. coli ATCC 25922.A slit was then cut into the plate with a sterile scalpel blade. Intothe slit, a suspension containing a heavy inoculum (≧10⁹ CFU permilliliter) of the test organism was dispensed by capillary action froma micropipette tip. A cefoxitin disk was then placed on the agar 3millimeters from the slit. After overnight incubation at 35° C., theplate was examined for evidence of cefoxitin inactivation, as indicatedby a characteristic distortion of the inhibition zone margin. Theabsence of this feature indicated no significant inactivation ofcefoxitin (FIG. 3).

AmpC Disk Test

Enzymatic inactivation of cefoxitin was also investigated by a disk testthat used a filter paper disk impregnated with a permeabilizing agent ofthe invention, TE, as described elsewhere herein. The disk wasrehydrated with 20 microliters of sterile saline and then inoculatedwith several colonies of the test organism. The surface of aMueller-Hinton agar plate was inoculated with a lawn of a standardizedinoculum of E. coli ATCC 25922. The inoculated disk was then placedbeside a cefoxitin disk (almost touching) on the inoculated plate. Afteran overnight incubation at 35 degrees Celsius, a positive test appearedas a flattening or indentation of the cefoxitin inhibition zone in thevicinity of the test disk. A negative test had an undistorted zone (FIG.4).

Isoelectric Focusing

Crude sonicates were subjected to analytical isoelectric focusing (IEF)on an ampholine polyacrylamide gel (pH range 3.5-9.5) on a flatbedapparatus (Multiphor LKB). Inhibitor overlays of the IEF gel wereperformed with 1000 μM clavulanic acid and cloxacillin.

Results

Thirty Klebsiella spp. isolates with cefoxitin MICs ≧16 μg/ml weretested with the AmpC disk test. Of these, 20 were negative and ten werepositive. The negative isolates were confirmed as negative with thethree-dimensional test. IEF and inhibitor studies revealed that nine ofthe ten positive isolates produced β-lactamases with pIs of either 7.2,7.7, or ≧9.0 that were inhibited by cloxacillin. These findings wereconsistent with AmpC production. These enzymes were interpreted to beplasmid-mediated because Klebsiella spp. lack a chromosomal AmpC. Theother positive isolate had an elevated imipenem MIC (64 μg/ml) andproduced a carbapenem-hydrolyzing enzyme that was also capable ofhydrolyzing cefoxitin.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1. A method of determining antibiotic susceptibility of a microorganismcomprising: a) admixing a test microorganism whose susceptibility is tobe determined with an antibiotic to which susceptibility is to beassayed, a permeabilizing agent for the test microorganism present in anon-growth-inhibiting microorganism-permeabilizing effective amount, anda strain of microorganism with known susceptibility for said antibioticto form an assay culture; b) providing a control comprising the testmicroorganism, the antibiotic, and said strain of microorganism, withouta non-growth-inhibiting microorganism-permeabilizing effective amount ofa permeabilizing agent; c) incubating said assay culture and controlunder appropriate conditions and for a time sufficient to determine thesusceptibility of the test microorganism to said antibiotic; and d)comparing growth in the assay culture to growth in the control, whereby,significantly more growth or a distortion in a zone of inhibition in theassay culture is indicative of resistance of the test microorganism tothe antibiotic attributable to inactivation of the antibiotic.
 2. Themethod of claim 1, wherein said permeabilizing agent is dissolved in ordispersed on a carrier.
 3. The method of claim 1, wherein thepermeabilizing agent is a buffer solution.
 4. The method of claim 3,wherein said buffer solution is Tris/EDTA.
 5. The method of claim 1,wherein the permeabilizing agent is a surfactant or detergent.
 6. Themethod of claim 2, wherein said carrier is a solid carrier.
 7. Themethod of claim 6, wherein said solid carrier is a paper disk.
 8. Themethod of claim 2, wherein said carrier is a liquid carrier.
 9. Themethod of claim 1, wherein said test microorganism and saidpermeabilizing agent are admixed before admixing with other componentsof the assay culture.
 10. The method of claim 1, wherein said assayculture is provided on solid growth medium.
 11. The method of claim 10,wherein said permeabilizing agent is provided on a solid carrier, saidtest microorganism is applied to said solid carrier, said antibiotic isprovided on a solid support, said strain of microorganism is inoculatedonto the solid growth medium; said admixing is by placing the solidcarrier proximal said solid support on the solid growth medium, and saidsignificant growth is observed as a distortion in a zone of inhibitionin the assay culture.
 12. The method of claim 1, wherein said antibioticis a beta-lactam-containing antibiotic.
 13. The method of claim 12,wherein said beta-lactam-containing antibiotic is a cefoxitin.
 14. Themethod of claim 12, wherein said beta-lactam-containing antibiotic is acarbapenem.
 15. The method of claim 1, wherein said assay culture isprovided in liquid growth medium.
 16. The method of claim 1, furthercomprising: detecting an antibiotic-inactivating factor produced by thetest microorganism based on the growth in the assay culture and control,whereby, significantly more growth or a distortion in a zone ofinhibition in the assay culture is indicative of production of anantibiotic-inactivating factor.
 17. The method of claim 16, wherein saidantibiotic-inactivating factor is a beta-lactamase.
 18. The method ofclaim 17, wherein said beta-lactamase is an AmpC.
 19. The method ofclaim 17, wherein said beta-lactamase is a carbapenemases.
 20. A methodof determining antibiotic susceptibility of a microorganism comprising:a) admixing a test microorganism whose susceptibility is to bedetermined with an antibiotic to which susceptibility is to be assayed,a permeabilizing agent for the test microorganism present in anon-growth-inhibiting microorganism-permeabilizing effective amount, anda strain of microorganism with known susceptibility for said antibioticto form an assay culture; b) incubating said assay culture underappropriate conditions and for a time sufficient to determine thesusceptibility of the test microorganism to said antibiotic; and c)detecting growth in the assay culture, whereby, growth in the assayculture is indicative of resistance of the test microorganism to theantibiotic.
 21. The method of claim 20, wherein said permeabilizingagent is dissolved in or dispersed on a carrier.
 22. The method of claim20, wherein the permeabilizing agent is a buffer solution.
 23. Themethod of claim 22, wherein said buffer solution is Tris/EDTA.
 24. Themethod of claim 20, wherein the permeabilizing agent is a surfactant ordetergent.
 25. The method of claim 21, wherein said carrier is a solidcarrier.
 26. The method of claim 25, wherein said solid carrier is apaper disk.
 27. The method of claim 21, wherein said carrier is a liquidcarrier.
 28. The method of claim 20, wherein said test microorganism andsaid permeabilizing agent are admixed before admixing with othercomponents of the assay culture.
 29. The method of claim 20, whereinsaid assay culture is provided on solid growth medium.
 30. The method ofclaim 29, wherein said permeabilizing agent is provided on a solidcarrier, said test microorganism is applied to said solid carrier, saidantibiotic is provided on a solid support, said strain of microorganismis inoculated onto the solid growth medium; said admixing is by placingthe solid carrier proximal said solid support on the solid growthmedium, and said significant growth is observed as a distortion in azone of inhibition in the assay culture.
 31. The method of claim 20,wherein said antibiotic is a beta-lactam-containing antibiotic.
 32. Themethod of claim 31, wherein said beta-lactam-containing antibiotic is acefoxitin.
 33. The method of claim 31, wherein saidbeta-lactam-containing antibiotic is a carbapenem.
 34. The method ofclaim 20, wherein said assay culture is provided in liquid growthmedium.
 35. The method of claim 20, further comprising: detecting anantibiotic-inactivating factor produced by the microorganism based onthe growth in the assay culture, wherein significant growth of thestrain of microorganism or a distortion in a zone of inhibition in theassay culture is indicative of production of an antibiotic-inactivatingfactor.
 36. The method of claim 35, wherein said antibiotic-inactivatingfactor is a beta lactamase.
 37. The method of claim 36, wherein saidbeta-lactamase is an AmpC.
 38. The method of claim 36, wherein saidbeta-lactamase is a carbapenemase.
 39. A kit for determining antibioticsusceptibility of a test microorganism comprising a permeabilizingagent-impregnated disk and instructions for performing the methodaccording to claim
 1. 40. A kit for determining antibioticsusceptibility of a test microorganism comprising a permeabilizingagent-impregnated disk and instructions for performing the methodaccording to claim
 20. 41. The method of claim 12 wherein saidbeta-lactam-containing antibiotic is an imipenem.
 42. The method ofclaim 31 wherein said beta-lactam-containing antibiotic is an imipenem.